PROJECT SUMMARY Mortality from myocardial infarction is decreasing; however, survivors are at high risk of developing ischemic cardiomyopathy (ICM). Understanding the mechanisms involved in that transition may help develop methods to prevent it. Ischemic damage produces a form of dyssynchronous contraction that cannot be treated with conventional therapies (Cardiac Resynchronization Therapy, CRT). However, the Principal Investigator previously discovered a critical molecular mechanism of CRT: it reactivates glycogen synthase kinase 3? (GSK-3?) and thus restores myofilament function. This proposal will leverage the molecular mechanism discovered in CRT in a patient population that cannot respond to it, ICM patients. Preliminary data reveals that human and mouse ICM samples exhibit myofilament calcium desensitization, and exogenous treatment with GSK-3? restores calcium sensitivity, suggesting the functional defect is linked to deactivation of GSK-3?. Further, new preliminary data has identified an independently regulated pool of GSK-3? localized to the myofilament that decreases significantly in human ICM, which correlates with the decrease in calcium sensitivity. Importantly, additional preliminary data suggest the localization of GSK-3? to the myofilament is mediated by phosphorylation of GSK-3? at tyrosine 216. This could allow targeted modulation of the myofilament pool of GSK-3? as a therapeutic strategy to improve myofilament function. Thus, based on these new preliminary data, this proposal addresses the central hypothesis that ischemia de-activates a myofilament pool of GSK-3? via altering tyrosine 216 (Y216) phosphorylation, decreasing phosphorylation of its myofilament targets and depressing myofilament function. There are three specific aims. Aim 1 will address the hypothesis that ICM decreases myofilament function in a GSK-3? dependent manner. Genetic mouse models that alter GSK-3? activity will be subjected to surgical induction of myocardial infarction to generate ICM and then myofilament function and GSK-3? activity will assayed. Human tissue from ICM patients will be studied similarly. Aim 2 will address the the hypothesis that phosphorylation at Y216 on GSK-3? modulates its binding to the myofilament and ICM decreases the amount and activity of GSK- 3? at the myofilament. Mutant forms of GSK-3? where the Y216 site is unphosphorylatable or mimic constitutive phosphorylation will be expressed in cardiac myocytes to determine where and how GSK-3? binds to the myofilament. Myofilament function will also be assessed to determine whether these mutant forms of GSK-3? can restore function in the GSK-3? knock-out mouse. The last aim will address the hypothesis that GSK-3? can normalize the myofilament phospho-proteome in ICM patients and ICM mouse tissue using state of the art mass spectrometry approaches. The long-term objective of this project is to identify the mechanisms by which GSK-3? affects myofilament function in the ICM heart, with the goal of discovering a therapeutic approach to prevent the transition to ischemic cardiomyopathy after a myocardial infarction.